The proposed research will concentrate on the mechanism of action of hormones on hepatic gluconeogenesis. The hypothesis to be tested is as follows: Hormonal control of hepatic gluconeogenesis is mediated by cAMP dependent and/or independent phosphorylation of cytoplasmic enzymes involved in the F6P/FDP and PEP/Pyruvate substrate cycles. The research has focussed on L-pyruvate kinase and FDPase as sites of hormonal control but we now propose to expand our efforts to phosphofructokinase (PFK). The effect of glucagon, insulin, and catecholamines on flux through PFK in intact rat hepatocytes will be characterized and correlated with studies on the effect of these hormones on 32P incorporation into PFK in intact hepatocytes. The labeled enzyme will be isolated by immunological methods. The phosphorylation state of the enzymes in intact cells will be correlated with enzyme activity measured in hepatocyte extracts and with cAMP levels. The protein kinase and phosphoprotein phosphatases which are presumably responsible for modification of these enzymes in liver will be isolated and characterized, particularly with regard to cAMP-dependency and their ability to alter the activity of the enzyme. Regulation of the kinases and phosphatases by hormones will also be studied. Ultimately these studies should allow us to determine if PFK is regulated by phosphorylation and if this regulation is cAMP-dependent or -independent in nature.